CN112076248A

CN112076248A - Application of perilla leaf extract in preparation of medicine for preventing and/or treating asthma

Info

Publication number: CN112076248A
Application number: CN201910516462.1A
Authority: CN
Inventors: 林明宝; 姚春所; 侯琦; 马培; 白金叶; 张梓倩; 吉腾飞; 范燕楠; 杨慧
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-06-14
Filing date: 2019-06-14
Publication date: 2020-12-15

Abstract

The invention discloses an application of perilla leaf extract in preparing a medicament for treating and/or preventing asthma. The perilla leaf extract disclosed by the invention has a reversing effect on lung inflammation pathological injury of an asthma model mouse; can obviously inhibit the generation of inflammatory cytokines TNF-alpha, IL-1 beta and the like in the alveolar lavage fluid of the model mouse; remarkably inhibiting the total number of leucocytes, the recruitment of lymphocytes and eosinophils in the alveolar lavage fluid of the model mouse; remarkably improves the asthma action of a model mouse.

Description

Application of perilla leaf extract in preparation of medicine for preventing and/or treating asthma

Technical Field

The invention relates to a perilla leaf extract, a preparation method thereof and application thereof in preparing a medicament or food for preventing and/or treating asthma, belonging to the technical field of medicines or foods.

Background

Bronchial asthma is one of the most common chronic respiratory diseases in the world at present, and researches show that about 3 hundred million asthma patients occupy about 3000 ten thousand in China, the morbidity is as high as 1-4%, and the health of the people is seriously harmed. The pathogenesis of asthma is complex, multiple mechanisms such as airway inflammation, allergic reaction, airway hyperresponsiveness, airway remodeling and the like are involved in the pathological process of asthma, and the airway inflammation is the disease basis of respiratory diseases such as asthma and the like, is the root cause of repeated occurrence of asthma symptoms, and plays an important decisive role in the occurrence, development and return of asthma. Therefore, the control of airway inflammation is an essential means for effectively treating asthma, and is one of the principles for clinical prevention and treatment of asthma. However, to date, there is no ideal drug for the treatment of airway inflammation. Researches find that the inhalation of the corticoids for treating airway inflammation has certain drug resistance, and the long-term use of the corticoids has more problems, thereby obviously increasing the occurrence risk of adverse reactions such as infectious pneumonia, growth retardation of children and the like. In addition, non-steroidal anti-inflammatory drugs are not suitable for clinical treatment of asthma due to their adverse reactions such as induction of severe asthma and enhancement of bronchoconstriction of the bronchi to stimulation with histamine, LTD4, etc. Therefore, the search for a safer and more effective new medicine for treating asthma airway inflammation becomes a hot point for the research of anti-inflammatory immune medicines at home and abroad.

In recent years, the research on the anti-inflammatory action of Chinese herbal medicines has greatly progressed, a lot of researches on asthma treated by traditional Chinese medicines are reported, the traditional Chinese medicine considers that 'phlegm obstruction of air passages, lung loss and descent, wind excess and phlegm obstruction, and air passage spasm are main pathogenesis of asthma', and the effects of clearing heat and removing toxicity, dispelling wind and removing dampness and the like of the traditional Chinese medicines are closely related to the anti-inflammatory immunity of modern medicine. Perilla (Perilla frutescens L.) is an annual herb of Perilla of Labiatae, and its leaf is a common clinical Chinese medicine, has pungent taste and warm nature, has effects of relieving exterior syndrome, dispelling pathogenic qi, regulating stomach function, etc., and can be used for treating common cold due to wind-cold, cough, emesis, vomiting of pregnancy, fish and crab poisoning, etc. The active ingredients of the perilla leaf, such as volatile oil, flavone, phenolic acid and the like, have good pharmacological activities of resisting inflammation, resisting oxidation, resisting allergy, stopping bleeding, reducing blood fat and the like. At present, no relevant research report about perilla leaf extract and asthma airway inflammation treatment by the perilla leaf extract is found, theoretical basis and practical experience are provided for effectively explaining the asthma treatment effect of traditional Chinese medicines and searching anti-asthma traditional Chinese medicines with better activity, the perilla leaf extract is prepared, and the anti-asthma airway inflammation activity of the perilla leaf extract is evaluated.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the application of the perilla leaf extract in preparing the anti-asthma medicine.

In order to solve the technical problem, the invention provides the following technical scheme:

the technical scheme of the invention provides application of a perilla leaf extract in preparing an anti-asthma medicine, which is characterized in that the preparation method of the perilla leaf extract comprises the following steps:

1) using perilla leaf as raw material, water or C₁～C₅Extracting with lower alcohol water solution as solvent, recovering water-containing lower alcohol solvent, and concentrating the recovered solution to obtain extract;

2) adding water into the extract for dispersion, adding a low-polarity organic solvent for extraction, and concentrating the residual water solution to obtain an extract;

3) the obtained extract is represented by formula C₁～C₅Precipitating with lower alcohol water solution, filtering, recovering water-containing lower alcohol solvent from the filtrate, drying, and pulverizing to obtain folium Perillae extract.

In step 1) of the above-mentioned production method,

the extraction solvent is water or C₁～C₅Aqueous lower alcohol solution, preferably C₁～C₃An aqueous lower alcohol solution, more preferably an aqueous ethanol solution;

C₁～C₅the lower alcohol concentration of the lower alcohol aqueous solution is 0 to 95% (v/v), preferably 50 to 80% (v/v), and more preferably 65 to 75% (v/v);

the extraction method comprises soaking, percolating, decocting or heating and refluxing, preferably decocting or heating and refluxing;

the extraction times are 1-5 times, preferably 2-3 times;

the concentration is normal pressure concentration or reduced pressure concentration, preferably reduced pressure concentration.

In step 2) of the above-mentioned production method,

the low-polarity organic solvent used for extraction is cyclohexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, n-butanol, isoamyl alcohol or a mixed solvent thereof, preferably ethyl acetate or n-butanol solvent, and more preferably n-butanol solvent;

the number of extractions is 1-5, preferably 2-3.

In step 3) of the above-mentioned production method,

alcohol precipitation with C₁～C₅The lower alcohol aqueous solution is preferably C₂～C₃An aqueous lower alcohol solution, more preferably an aqueous ethanol solution;

C₁～C₅the lower alcohol concentration is 50 to 90% (v/v), preferably 60 to 85% (v/v), and more preferably 75 to 85% (v/v);

the alcohol precipitation time is 8-72h, preferably 12-24 h;

the drying method includes normal pressure drying, vacuum drying, freeze drying, and spray drying, preferably vacuum drying and freeze drying.

For OVA-induced allergic asthma in mice, Perilla leaf extract is administered by intragastric administration at dosage of 50 mg/kg and 100mg/kg, and has inhibitory effect on the generation of inflammatory cytokines TNF-alpha and IL-1 beta in mouse BALF; inhibiting leukocyte total number, lymphocyte and eosinophil recruitment in BALF; has reversing effect on pathological changes of mouse lung inflammation.

The second aspect of the technical scheme of the invention provides application of a pharmaceutical composition in preparing an anti-asthmatic medicament, wherein the pharmaceutical composition contains the perilla leaf extract in the first aspect and a pharmaceutically acceptable carrier or excipient. Wherein the perilla leaf extract is prepared as described in the first aspect of the present invention.

For this purpose, the active ingredient may, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants in a suitable administration form or dosage form for human administration.

The pharmaceutical compositions of the present invention may be administered in unit dosage form, either enterally or parenterally, for example orally, intramuscularly, subcutaneously, nasally, oromucosally, dermally, peritoneally or rectally, and the like.

The route of administration of the pharmaceutical composition of the present invention may be administration by injection. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc. The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The composition can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, ethylparaben, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium hydrogen carbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

For making the administration units into pills, a wide variety of carriers well known in the art can be used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc.

For making the administration unit into a suppository, various carriers well known in the art can be widely used. As examples of the carrier, there are, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, higher alcohol enzymes, gelatin, semisynthetic glycerase and the like.

To encapsulate the administration units, the active ingredient is mixed with the various carriers described above, and the mixture thus obtained is placed in hard gelatin capsules or soft gelatin capsules. Or making into microcapsule, suspending in aqueous medium to form suspension, or making into hard capsule or injection.

For example, the composition of the present invention is formulated into an injectable preparation, such as a solution, a suspension solution, an emulsion, a lyophilized powder, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, etc. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. These adjuvants are commonly used in the art.

In addition, if desired, colorants, preservatives, flavors, flavorings, sweeteners, or other materials may also be added to the pharmaceutical preparation.

The dose of the pharmaceutical composition of the present invention to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, the number of administrations, etc., and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The amount of the actual effective drug contained in the final formulation of the pharmaceutical composition of the present invention may be suitably adjusted to achieve the desired therapeutically effective amount thereof, to accomplish the purpose of the present invention for treating asthma. In general, the daily dose of the extract is 0.001mg/kg body weight to 500mg/kg body weight, preferably 3mg/kg body weight to 30mg/kg body weight, for a patient weighing about 75 kg. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms, which is limited by the clinical experience of the administering physician and the dosage regimen.

The invention has the advantages that: (1) the invention belongs to a traditional Chinese medicine preparation, and has pure natural components and low toxic and side effects; (2) the invention can effectively prevent and treat asthma; (3) the product has simple preparation process and is easy for standardized production.

Drawings

FIG. 1 Effect of perilla leaf extract on pathological changes in lung tissue in asthmatic mice;

Detailed Description

The present invention is further illustrated by the following examples, but the present invention is not limited to these examples.

Example 1: preparation method of folium Perillae extract

Taking 1.5kg of dried perilla leaf, adding 5 liters of 70% ethanol, heating and refluxing for 1.5 hours, and filtering while the solution is hot. The filter residue is treated again by the same method; mixing the filtrates, and recovering ethanol under reduced pressure to obtain 65g extract. 300mL of water is added into the extract, the mixture is stirred to prepare suspension, and the suspension is extracted for 2 times by 150mL of ethyl acetate respectively. Dissolving the rest water part with 1580mL of 95% ethanol, precipitating at room temperature for 24h, vacuum filtering, recovering ethanol from the filtrate under reduced pressure, freeze drying, and pulverizing to obtain folium Perillae extract.

Example 2: preparation of dosage forms

And (3) capsule preparation: 100g of perilla leaf extract, 147.5g of dextrin and 2.5g of magnesium stearate are added, and after fully and uniformly stirring, 1000 granules are prepared according to a capsule preparation method and are filled into 250mg capsules, wherein each granule contains 100mg of perilla leaf extract, and the total amount of the extract is not less than 50%.

And (3) tablet preparation: taking 100g of perilla leaf extract, adding 100g of starch, 80g of dextrin and 15g of sucrose, fully stirring and uniformly mixing, preparing into granules, drying at the temperature of below 60 ℃, adding a proper amount of magnesium stearate and talcum powder, uniformly mixing, pressing into 1000 tablets, and coating sugar coat or film coat to obtain the finished product.

Granules: taking 100g of perilla leaf extract, adding a proper amount of sucrose and dextrin, preparing into granules according to a granule preparation method, and drying to prepare 1000 g.

Oral liquid: extracting folium Perillae with 70% ethanol under reflux for 1.5 hr for 2 times, filtering, mixing filtrates, and recovering ethanol under reduced pressure; adding equal amount of water into the extract, stirring to obtain suspension, and extracting with ethyl acetate for 2 times; dissolving water in 95% ethanol, precipitating at room temperature for 24 hr, vacuum filtering, adjusting pH to 7.0 with 40% sodium hydroxide, stirring, refrigerating at 4-8 deg.C for 48 hr, filtering, adding sucrose 300g into the filtrate, stirring to dissolve, adding essence, adjusting pH to 7.0, adding water to 1000ml, stirring, standing for 12 hr, filtering, bottling, and sterilizing.

Pharmacological experiments

Experimental example 1: inhibitory effect of perilla leaf extract on OVA-induced allergic asthma in mice

The method comprises the following steps: SPF male BALB/c mice, 18-20g, were randomly divided into a normal control group, a model control group, a positive control group (dexamethasone, 0.5mg/kg, gavage), and a drug treatment group (perilla leaf extract 50,100mg/kg, gavage), each group containing 20 mice. On the 1 st day of experiment, animals in each group were intraperitoneally injected with 100 μ L (20 μ g) of OVA solution and 100 μ L of aluminum hydroxide gel, and a blank control group was administered with an equal volume of physiological saline; and injecting OVA and aluminum hydroxide gel with the same sensitizing dose into the abdominal cavity for immune enhancement at 7 days and 14 days after the first sensitization respectively. Animals were gavaged 1 time a day for 14 consecutive days after the 14 th boost. On day 26 after the first sensitization, mice were anesthetized, and challenged with a 1.60mg/ml OVA solution of 50. mu.L (80. mu.g) by tracheal instillation 1.60mg/ml for 3 consecutive days, except for the placebo group, which was given an equal volume of vehicle. After 24h of the last challenge, mice were anesthetized with phenobarbital sodium, perfused with 0.7mL of pre-cooled normal saline, washed 3 times, aspirated with bronchoalveolar lavage fluid (BALF), centrifuged, and the supernatant was used to analyze the contents of TNF- α and IL-1 β; resuspending the cells in PBS buffer solution, and performing differential counting on the white blood cells; lung tissue was taken for histopathological examination.

As a result:

(1) perilla leaf extract for improving injury of lung tissue inflammatory pathology of OVA-induced allergic asthma mice

The pathological staining results are shown in fig. 1, and the pathological scores are shown in table 1. Compared with the blank control group, the mice in the model group can see obvious inflammatory cell infiltration (mainly eosinophils, lymphocytes, neutrophils and mononuclear macrophages) around the bronchus and the blood vessels, the bronchial epithelial cells are exfoliated, villous hyperplasia, goblet cell metaplasia, increase of viscous secretion, moderate broadening of local alveolar space, reduction of alveolar space and fibrin exudation in the alveolar space.

As a result: perilla leaf extract can be administered by intragastric administration at a dose of 50,100mg/kg, and can remarkably reduce inflammatory cell infiltration and epithelial cell hyperplasia around bronchus and blood vessel, and has remarkable therapeutic effect on OVA-induced allergic asthma mice.

Table 1 results of scoring the inflammatory lesions of lung tissue pathology in asthmatic mice by perilla leaf extract (Mean ± Std, n ═ 4)

Remarking: compared with the blank control group, the # p is less than 0.05, and the # p is less than 0.01; p < 0.05, p < 0.01, compared to model control

(2) Inhibition of inflammatory cell recruitment in alveolar lavage fluid (BALF) in asthmatic mice by perilla leaf extract

Influence of Perilla leaf extract on the Total leukocyte count in BALF of asthmatic mice

The airway of asthma patients is infiltrated by neutrophils, eosinophils, basophils, etc. In asthma, leukocytes aggregate in the airways, synthesize and secrete a variety of cytokines that dominate the inflammatory response and play important roles in the progression of asthma. The experiment aims at investigating the effect of perilla leaf extract on total inflammatory cell recruitment in BALF of asthmatic mice, and the results are shown in Table 2.

As a result: perilla leaf extract was administered intragastrically at a dose of 50,100mg/kg to asthma model mice to reduce leukocyte recruitment in BALF of the model mice.

Table 2 effect of perilla leaf extract on the total number of leukocytes in BALF of asthmatic mice (Mean ± Std n ═ 10)

② influence of perilla leaf extract on lymphocyte classification and counting in asthma mouse BALF

Allergic diseases are essentially caused by an abnormal balance of two subsets of lymphocytes (Th1 and Th2), predominant differentiation of Th2 cells and excessive expression of Th2 cytokines. The CD4+ T lymphocyte subgroup, regulatory T cells (Treg), plays an important role in maintaining the immune balance of the body, inhibits the generation of macrophages and inflammatory cytokines by secreting cytokines such as IL-10, TGF-beta and the like, mediates the tolerance of the body to allergens, inhibits Th2 type immune response, relieves asthma airway inflammation, and finally plays a role in inhibiting the development of asthma. Th17 lymphocyte and inflammatory mediator secreted by the same can promote the accumulation of neutrophil, eosinophil and inflammatory mediator in the airway of an asthmatic mouse, aggravate airway inflammation and are closely related to asthma airway remodeling. The experiment aims at investigating the influence of perilla leaf extract on lymphocyte recruitment in BALF of asthmatic mice, and the results are shown in Table 3.

As a result: perilla leaf extract can be administered by gavage to mice in allergic asthma model at a dose of 50,100mg/kg, to reduce lymphocyte recruitment in BALF of mice.

Table 3 effect of perilla leaf extract on lymphocyte differential count in BALF of asthmatic mice (Mean ± Std, n ═ 10)

③ influence of perilla leaf extract on eosinophil differential count in asthma mouse BALF

The aggregation and activation of eosinophils play a critical role in the respiratory inflammation of asthma, the cytoplasm of which contains a number of distinct protein granules, mainly the Eosinophilic Cationic Protein (ECP), the basophilic protein (MBP), the Eosinophilic Peroxidase (EPO), all of which are strongly cytotoxic, and many of the pathophysiological changes in asthma are due to the release of these proteins by eosinophils. Eosinophils degranulate and release active inflammatory mediators, causing bronchospasm, vascular congestion and increased permeability, tissue inflammation, leading to an asthmatic response. The experiment was aimed at investigating the effect of perilla leaf extract on eosinophil recruitment in BALF of asthmatic mice, the results are shown in table 4.

As a result: the perilla leaf extract can reduce eosinophilic granulocyte recruitment in BALF of mice when administered by gavage to allergic asthma model mice at a dose of 50 mg/kg and 100 mg/kg.

TABLE 4 Effect of perilla leaf extract on eosinophil differential count in BALF of asthmatic mice (Mean. + -. Std, n ═ 10)

(3) Inhibitory effect of perilla leaf extract on inflammatory factor production in alveolar lavage fluid (BALF) of asthmatic mice

Influence of perilla leaf extract on TNF-alpha generation in BALF of asthmatic mice

TNF-alpha has a wide range of pathophysiological roles in the development of asthma. The increase of TNF-alpha can activate vascular endothelial cells, increase vascular permeability, induce the expression of adhesion molecules among endothelial cells, enable inflammatory cells to adhere to the vascular endothelial cells, permeate into airway mucosal tissues from blood circulation and cause inflammatory reaction. The activated inflammatory cells release inflammatory mediators, further chemotactic eosinophils to release inflammatory mediators, cause the shedding of airway epithelial cells, promote the formation of airway epithelial injury and airway hyperreactivity, further cause airway spasm, increase of vascular permeability, edema of airway mucosa and the like, and induce or aggravate asthma. The experiment aims at investigating the influence of perilla leaf extract on TNF-alpha generation in BALF of asthmatic mice, and the results are shown in Table 5.

As a result: perilla leaf extract can be administered by intragastric administration to mice with allergic asthma model at a dose of 50 mg/kg and 100mg/kg to reduce TNF-alpha production in mice BALF.

TABLE 5 Effect of Perilla extract on TNF-. alpha.production in BALF of asthmatic mice (Mean. + -. Std n ═ 10)

② influence of Perilla leaf extract on IL-1 beta production in asthma mouse BALF

IL-1 beta is an endothelial cell activating factor and has obvious proinflammatory effect. IL-1 beta can induce airway eosinophilic infiltration, airway hyperresponsiveness and goblet cell proliferation, stimulate airway endothelial cells to release fibroblast growth factors, lead fibroblast proliferation and collagen synthesis, and participate in airway remodeling. IL-1 β promotes inter-alveolar rupture of the elastic fibers, increases pleural thickness and mucus production, and can cause fibrosis of the airway wall, thereby playing a role in airway remodeling during the pathophysiological process of asthma. The experiment aims at investigating the influence of perilla leaf extract on IL-1 beta production in BALF of asthmatic mice, and the results are shown in Table 6.

As a result: the folium Perillae extract can be administered to mice with allergic asthma model by intragastric administration at a dose of 50 mg/kg and 100mg/kg to reduce IL-1 beta production in BALF of mice.

TABLE 6 Effect of perilla leaf extract on IL-1. beta. production in BALF of asthmatic mice (Mean. + -. Std, n ═ 10)

Remarking: compared with the blank control group, the # p is less than 0.05, and the # p is less than 0.01; p < 0.05, p < 0.01 compared to model control.

Claims

1. An application of a perilla leaf extract in preparing a medicine for preventing and/or treating asthma is characterized in that the preparation method of the perilla leaf extract comprises the following steps:

2) adding water into the extract obtained in the step 1) for dispersion, adding a low-polarity organic solvent for extraction, and concentrating the residual aqueous solution to obtain an extract;

3) mixing the extract obtained in step 2) with C₁～C₅Precipitating with lower alcohol water solution, filtering, recovering water-containing lower alcohol solvent, concentrating the recovered solution, drying, and pulverizing to obtain folium Perillae extract.

2. The use according to claim 1, wherein the extraction method in step 1) is impregnation, percolation, decoction or heating reflux.

3. Use according to claim 1, characterized in that in step 1) C₁～C₅The lower alcohol concentration of the lower alcohol aqueous solution is 0-95%.

4. The use according to claim 1, wherein in the step 2), the low-polarity organic solvent used for extraction is cyclohexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, n-butanol, isoamyl alcohol or a mixed solvent thereof; the extraction times are 1-5 times.

5. Use according to claim 1, characterized in that in step 3) C₁～C₅The lower alcohol concentration of the lower alcohol aqueous solution is 50-90%.